Environmental Protection Agency. Alternate Test Procedure Validation Report. for Enzymatic Reduction Method of Nitrate-N Analysis

Transcription

1 Environmental Protection Agency Alternate Test Procedure Validation Report for Enzymatic Reduction Method of Nitrate-N Analysis Compiled and Written by Wilbur H. (Bill) Campbell and Ellen R. Campbell The Nitrate Elimination Co., Inc. (NECi) 334 Hecla St. Lake Linden, MI Revised: 20 February 2014

2 NECi ATP Validation Study Report 3.0 Acknowledgments: Charles J. Patton, Methods R&D Program, U.S. Geological Survey, National Water Quality Laboratory, P.O. Box 25585, Lakewood, CO , and Dr. William Lipps, formerly of OI Analytical Co., are thanked for their assistance with the design of the Inter-Laboratory ATP Validation Study and preparation of the Validation Study Report. All the laboratories, which are listed in Table 2, are thanked for participating in the Inter-Laboratory ATP Validation Study. The following U. S. departments and agencies are thanked for providing funding for research and development studies carried out at NECi over the past 20 years: Department of Energy (DOE), Environmental Protection Agency (EPA), National Institutes of Health (NIH), National Science Foundation (NSF), and Department of Agriculture (USDA). The State of Michigan is thanked for providing funding to NECi via Michigan Emerging Technology Fund of the Michigan Economic Development Corporation. NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page ii

3 NECi ATP Validation Study Report 3.0 Table of Contents Page Report 1 Section 1 Introduction 2 Section 2 Inter-Laboratory Validation Study 3 Section 2.1 Study Objectives and Plan 3 Section 2.2 Sources of Sample Matrices and Handling of the Samples 3 Section 2.3 Participating Laboratories and Analytical Equipment 4 Section 3 Validation Study Results and Discussion 4 Section 3.1 Summary of Quality Control 5 Section 3.2 Nitrate-N Content and Spike Analysis of Sample Wastewater Matrices 7 Section 3.3 Evaluation of Accuracy for the Method 7 Section 3.4 Comparison of Enzymatic Reduction and Cadmium Reduction Methods 8 Section 4 Validation Conclusions 9 Section 5 References and Glossary 11 Tables Table 1 List of Sample Matrices Table 2 List of Participating Laboratories Table 3 Calibration of Enzymatic Reduction Method Summary Table 4 Enzymatic Reduction Efficiency Summary Table 5 Initial Performance and Recovery (IPR) Summary Table 6 Ongoing Performance and Recovery (OPR) Summary Table 7 Minimum Detection Limit (MDL) Summary Table 8 Nitrate-N Content and Spike Analysis of Sample WastewaterWastewater Matrices Table 9 Detailed Data Supporting Table 8 Table 10 Standard Reference Materials Summary Table 11 Comparison of Nitrate-N Content for the Sample Matrices Determined by Nitrate Reductase Method, and by Cadmium Reduction Method Appendices Appendices are not included contact NECi if needed Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Inter-Laboratory Validation Study Plan Description of the Enzymatic Reduction Method by Discrete Analyzer FACET Protocol for Sample Handling and Shipping Certification Sheets for Nitrate and Nitrite Standards utilized in this study Summary Data Sheets and Bench Sheets for Each Laboratory (Excel Files) Summary Data Sheets for MDL (Excel Files) NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page iii

4 Report NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 1

5 Section 1 Introduction This Validation Study Report supports development of Nitrate plus Nitrite Nitrogen by Nitrate Reductase (Campbell, et al., 2006), also called Enzymatic Reduction Method for Nitrate-N Analysis, for use as an USEPA Alternate Test Procedure (ATP) for Tier 3 level (nationwide) determination of nitrate plus nitrite nitrogen in wastewater, and other aqueous solutions (EPA, 1999). The enzyme Nitrate Reductase (EC , NaR) catalyzes the reduction of nitrate to nitrite with the reducing power provided by the natural reductant, reduced Nicotinamide Adenine Dinucleotide (NADH), which is a thermodynamically irreversible reaction. Nitrate + NADH + H + Nitrite + NAD + + H 2 O This is a green, non-toxic method for nitrate-n analysis. Eukaryotic NaR is a complex enzyme which contains a polypeptide chain of more than 900 amino acid residues and two metal ions (Fe 3+ and Mo 6+ ) and three organic cofactors (Flavin Adenine Dinucleotide (FAD), Heme, and Molybdopterin) (Campbell, 1999). Since the natural NaR is of low abundance in plants, algae and fungi, recombinant DNA technology is utilized to produce the enzyme from a plant NaR gene (Arabidopsis thaliana) in the yeast Pichia pastoris which is designated AtNaR2 (Campbell et al., 2006). Recombinant AtNaR2 is purified from the yeast extract to near homogeneity using immobilized metal ion affinity chromatography via the Histidine-tag built into the recombinant gene product. The purified AtNaR2 is highly stable and can be stored in a buffered solution at -80 C indefinitely. Furthermore, when the AtNaR2 is freeze-dried and stored, dry and under vacuum in an opaque package, it can be shipped at room temperature and will remain stable for up to 6 months. NaR-based Nitrate-N analysis is formulated as a method with a small volume, which is ideal for modern instruments such as the automated discrete analyzer. The method s formulation consists of a biochemical buffer to maintain ph near neutrality, the reconstituted NaR (stable for 18 hours), a precise solution of NADH, and the small volume of sample to be analyzed for Nitrate- N content. For example, in the discrete analyzer, the volume of buffered AtNaR2 is 55 µl, NADH 12 µl, and sample 5 µl (Patton and Kryskalla, 2011; 2013). Compared to EPA method 353.2, where the sample is often 20 ml, the enzymatic reduction method has obvious advantages in sample and waste handling. After the reduction of nitrate to nitrite is complete, requiring about 10 min, the nitrite is determined colorimetrically as in EPA method 353.2, which involves the sequential addition of sulfanilamide (SAN) and N (1 Naphthyl)ethylenediamine dihydrochloride (NED) and measurement at 540 ± 20 nm. NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 2

6 Section 2 Inter-Laboratory Validation Study The details of the Inter-Laboratory Validation Study are presented in this section. The Nitrate Elimination Co., Inc. (NECi), Lake Linden, Michigan, coordinated the Inter-Laboratory Validation Study of the Enzymatic Reduction Method based on Nitrate Reductase for determination of nitrate-n and nitrate/nitrite-n. In this study the Enzymatic Reduction Method for nitrate-n is not directly compared to the Cadmium Reduction Method for nitrate-n (EPA Reference Method 353.2), since the automated discrete analyzers used by the participating laboratories do not run the Reference Method. However, one participating laboratory analyzed the study s sample matrices using the Cd Reduction Method running on a continuous flow analyzer and this provided a comparative data set. In addition, two published studies have compared the Enzymatic Reduction Method to the Cd Reduction Method and demonstrated the equivalence of the two methods for determining nitrate-n and nitrate/nitrite-n in aqueous samples (Patton and Kryskalla, 2011; Patton and Kryskalla, 2013). Section 2.1 Study Objective and Design Plan The objective of the Inter-Laboratory Study of Nitrate-N Analysis by the Enzymatic Reduction Method based on Nitrate Reductase was to demonstrate the validity of the Method according to the Design Plan presented in Appendix A. The Design Plan was developed in accordance with ATP Protocol for Organic and Inorganic Analytes (USEPA, 1999) in order to validate the Method for Tier 3 (nationwide) status for both Drinking and Wastewater. The Study Plan was approved by Lemuel (Lem) Walker, Jr., Clean Water Act ATP Coordinator, U.S. Environmental Protection Agency, Office of Science and Technology, Engineering and Analysis Division (EAD) on 14June2013 (see Appendix A). Subsequently, the scope of the Inter-Laboratory Study was narrowed to just the Wastewater and Seawater Matrices, but the Study Plan was not altered to reflect this change. The Sample Matrices analyzed in the Inter-Laboratory Validation Study are listed in Table 1. The Enzymatic Reduction Method based on Nitrate Reductase is described according to EPA format in Appendix B, which was incorporated in the Design Plan. This Method was implemented by the Participating Laboratories which are listed in Table 2. The variety of wastewater matrices to be analyzed brings the study into compliance with the Clean Water Act (CWA). Section 2.2 Sources of Sample Matrices and Handling of the Samples The list of Sample Matrices is presented in Table 1. All matrices were collected on site and processed by filtration and preservation by addition of 4.5 Normal sulfuric acid to lower the ph to below 2 and cooled to <6 C, according to the Design Plan. The preservation status of the sample matrices is shown (Table 1). NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 3

7 The sample matrices were shipped in bulk to FACET Analytical Services and Technology, LLC, Indianapolis, IN FACET LLC filtered, acidified and aliquoted the sample matrices to certified clean bottles according to the Study Plan and EPA protocols (Appendix C). Each sample Matrix bottle was labeled with the designated matrix identification (i.e. WW-1, WW-2, etc.). Certified Nitrate Standards and Calibrants were purchased from ERA in individual, labeled bottles such that there was no handling of these until they reached the participating Laboratories. The packaged and sealed Sample Matrices and Nitrate Standards were shipped on blue ice to the Participating Laboratories according to the plan developed by FACET and approved by NECi as shown in Appendix C. According to the Design Plan, once the Sample Matrices were shipped, the Participating Laboratories were to store them at 4 C and they had to complete the analysis within 30 days. The deadline for analyses completion was July 17, Certification documents for the calibrants and nitrate standards are provided in Appendix D. Each laboratory provided their own nitrite standard and the certification for the Nitrite Standard utilized by one laboratory is also included in Appendix D. Section 2.3 Participating Laboratories and Analytical Equipment The list of Participating Laboratories is presented in Table 2. A more detailed list of information on the Participating Laboratories is presented in Table 2 in the Study Plan (Appendix A). Laboratory 6 dropped out of the study after the samples had been shipped to them and did not complete the analysis of the samples. All the Participating Laboratories were equipped with automated discrete analyzers (DA). Reagents for the Nitrate analysis, including the enzyme Nitrate Reductase (AtNaR2), were supplied to all laboratories by NECi. The enzyme (AtNaR2) consisted of a vacuum sealed, opaque pouch with a desiccant and a vial of freeze-dried AtNaR2 containing one unit of enzyme activity, where the unit of enzyme activity is defined as the amount of enzyme catalyzing the NADH-driven reduction of 1 µmol of nitrate to nitrite per min at 30 C and ph 7.5. AtNaR2 when stored in this form at room temperature (~25 C) is stable for up to six months. Each laboratory reconstituted the enzyme in phosphate buffer, ph 7.5, at the time of analysis according to instructions provided with the enzyme packet. For each type of DA being used in the study, a specific set of Standard Operating Procedures (SOP) has been developed which condenses the Method in Appendix B. As an example, the SOP for the OI Analytical DA has been added to the Method in Appendix B. Section 3 Validation Study Results and Discussion The results from the Inter-Laboratory Validation Study are contained in the Excel files in Appendix E and F. The original Excel files have been provided on a flash drive, which NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 4

8 accompanied the original version of this Report and have not been included with the current version of this report. Included within the Summary Excel file are the bench sheets where possible and, in some cases, as PDF or other files in the directory for each laboratory in the previously provided data sets. An explanation table for error codes used in the KoneLab AquaKem DA raw data files is provided in Appendix F. Supplemental Excel File for the MDL Study by Laboratory 5 is provided in Appendix F, which is supplied by attachment. For this section of the Report, summary tables have been prepared from the original and supplemental data in the Excel Summary Data Sheet files Appendix E and F). Section 3.1 Summary of Quality Control Statistical analysis of Calibration Curves reported by each laboratory are summarized in Table 3A. In many cases, the laboratories ran more than one standard curve and the summary in Table 3 represents selected statistical data. In all cases, the correlation coefficient (r 2 ) = or greater, except for Lab 7 and 10. However, both of these labs used a polynomial fit to the calibration results and found r 2 = and , for labs 7 and 10, respectively. In each case, the parameters for the slope and intercept are utilized to generate an equation relating the 540 nm (or 550 nm) to the Nitrate-N content of the unknown sample, such that the concentration of Nitrate-N (mg N/L) can be calculated from the Absorbance. In cases where a polynomial equation is used to described the relationship of Absorbance to Nitrate-N content, the slope is replaced by constants A and B, which are not listed in the table. Table 3B catalogs the nitrate concentrations of the standards used in the calibration. This information is useful in comparing the results from different labs, since some labs used as many as 8 calibration concentrations, while some used as few as 5 calibration concentrations. In addition, Lab 12 used 0.10 mg N/L as the lowest calibrant, while most of the other labs using a discrete analyzer used 0.05 mg N/L (Lab 7 used 0.04 mg N/L). Enzymatic reduction efficiencies for each laboratory are summarized in Table 4. All labs found enzymatic reduction efficiency of 95 % or greater. This establishes the effectiveness of the nitrate reductase-catalyzed enzymatic reduction of nitrate to nitrite under the conditions of the analytical method. Initial Performance and Recovery (IPR) and Ongoing Performance and Recovery (OPR) results from all laboratories are summarized in Table 5 and 6, respectively. IPR certified standard nitrate concentrations were 2.00, 2.50, and 2.70 mg N/L (see Appendix D). IPR mean recoveries for the selected data in Table 5 were from 96 to 106%, which meets the acceptance criterion of 100 ± 10%. Other IPR analysis by the labs, which are not shown in Table 5, also fell within the acceptance criteria of 90 to 110% recovery (Appendix E - Excel files). OPR certified standard nitrate concentrations were 2.00, 2.50, and 2.60 mg N/L (see Appendix D). OPR mean NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 5

9 recoveries for the selected data in Table 6 were from 96 to 104% which meets the acceptance criterion of 100 ± 10%. All other OPR recoveries, which are not shown in Table 6, were within the acceptable range of 90 to 110% recovery(appendix E - Excel files). The IPR and OPR results provide measures of the precision and accuracy of the Enzymatic Reduction Method of Nitrate-N Analysis. For IPR, five labs used the ERA 2.00 ± 0.02 mg N/L certified nitrate standard and obtained the mean value = ± 0.048; RSD = Four labs used an 2.50 mg N/L certified nitrate standard and obtained the mean value = ± mg N/L; RSD = % and. Only one lab used the 2.70 mg N/L certified nitrate standard and found the mean value = ± mg N/L; RSD = %. Thus, there was less than a 2% difference between the values determined and the known values of the certified nitrate standards. The OPR values yielded RSD from 0.52 to 5.66 % (Table 6). Thus, the Enzymatic Reduction Method is highly precise and accurate based on these IPR and OPR results. The final Quality Control evaluation is the determination of Minimum Detection Limit (MDL) for each laboratory s equipment; these results are summarized in Table 7. All the MDL values are below the level of the lowest calibrant for these analyzers (Table 3B), which indicates that the calibration curve for these analyzers is completely valid with respect to detecting nitrate-n at the lowest level of the calibration. Three published studies evaluated the MDL for Enzymatic Reduction Method for Nitrate-N Analysis (Patton et al., 2002; Patton and Kryskalla, 2011; Patton and Kryskalla, 2013). When the Method was run on an Air-segmented Continuous Flow Analyzer (Patton et al., 2002), the MDL was reported to be mg N/L. When the Method was run on a Discrete Analyzer (Patton and Kryskalla, 2011), the MDL was reported to be 0.02 mg N/L. When the MDL of the reference method, EPA Method 353.2, was determined on an Air-segmented Continuous Flow Analyzer (Patton et al., 2002; Patton and Kryskalla, 2013), it was found to be mg N/L. The lower MDL for the reference method is apparently due to differences in the analyzer equipment: Air-segmented Continuous Flow Analyzer uses the same cuvette for analyzing all samples and blanks; and the Discrete Analyzer uses a different cuvette for every sample and blank. Indeed, the Enzymatic Reduction Method for Nitrate-N Analysis, has a lower MDL for the Air-segmented Continuous Flow Analyzer than the Discrete Analyzer. Although the Discrete Analyzer uses a correction for background absorbance, it apparently does not correct for all the differences between the cuvettes (Patton and Kryskalla, 2011). Section 3.2 Nitrate-N Content and Spike Analysis of Wastewater and Seawater Matrices The Wastewater Matrices were analyzed for Nitrate-N content and spiked with 0.5 mg N/L and analyzed 6 times. These results are summarized in Table 8 and more detailed presentation of these results are presented in Table 9. Seawater was analyzed for Nitrate-N content and spiked NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 6

10 with 0.5 mg N/L and analyzed 6 times. These results are also summarized in Table 8 and 9. The Nitrate-N content of the matrices varied greatly and some had to be diluted before analysis. The sample matrices which were diluted to bring them within range were: WW-2, WW-5, WW-6, and WW-8. Relative Standard Deviations were less than 10%, except for three matrices. These three matrices with Relative Standard Deviations greater than 10% were ones that had Nitrate-N content less than the lowest calibrant of the Standard Curves: WW-1; WW-7; and SW-1. Nitrate-N content determined below the lowest calibrant of the Standard Curves may not be valid and would be expected to have greater error in the determination than ones falling within the range of the standard curves. For the diluted matrices, the spiking was carried out with the diluted matrix. The acceptance criterion for spike recovery in the Wastewater and Seawater matrices was 77 to 121%. The acceptance criterion for the Relative Percent Difference (RPD) of the concentration results of the Matrix Spike (MS) and Matrix Spike Duplicate (MSD) pair was 22%.As shown in Table 8 and 9, all of the spiking results yielded data within the acceptance criteria for mean spike recovery and RPD. The mean spike recoveries varied from 85.0 to 112.4%. The RPD varied from 0.08 to 9.6%. No data from Laboratory 8 is in Table 8 or 9, since the spiking protocol specified in the Study Plan was not followed. Specifically, this laboratory did not spike samples at the specified concentration level for all pairs, resulting in data that were not comparable to those generated by the other laboratories. Overall, none of the Wastewater Matrices, nor the Seawater Matrix, yielded a matrix effect on nitrate-n analysis by the Enzymatic Reduction Method, which indicates the method is not biased by the constituents of the sample matrices listed in Table 1. It is noteworthy that Nitrate-N content was successfully determined in seawater by the DA Enzymatic Reduction Method; however, the seawater matrix had such a low Nitrate-N content that the results were not valid, but the 0.5 mg N/L spikes were accurately determined. Thus, the salt content of seawater does not bias the Method even when no salt is included in the calibrants. A previous study of Nitrate- N determination in seawater by the Enzymatic Reduction Method also found that valid results were found compared to the cadmium reduction method; however, the calibrants in this study were prepared in synthetic seawater (Ringuet et al., 2011). 3.3 Evaluation of the Accuracy of the Method There were three reference standards analyzed by the participating laboratories in this study (Table 10). The first standard was SR-1 (ERA #608) and it was analyzed by 6 laboratories yielding a total of 38 analyses. The mean Nitrate-N content of SR-1 was ± mg N/L (RSD = %), which compared to the certified target value = 6.80 ± 0.12 mg N/L. The mean recovery of SR-1 was %. The two other reference standards were from the USGS (SRM-1 and SRM-2). SRM-1 was analyzed by 7 laboratories for a total 36 analyses and the NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 7

11 mean Nitrate-N content found was ± mg N/L (RSD = %). The target value was the Most Probable Value found by multiple laboratory analysis and reported on the USGS website (see Table 9 footnote for the website URL; also see Appendix D for the USGS SRM1 data) was ± mg N/L (RSD = %). The mean recovery of SRM-1 was %. SRM-2 was analyzed by 7 laboratories for a total 25 analyses and the mean Nitrate- N content found was ± mg N/L (RSD = %). The target value was the Most Probable Value found by multiple laboratory analysis and reported on the USGS website (see Table 9 footnote for the website URL; also see Appendix D for the USGS SRM2 data) was ± mg N/L (RSD = %). The mean recovery of SRM-1 was %. From the results of the analysis of the 3 standard references (Table 9) and the certified nitrate standards analyzed for Quality Control (see section 3.1), it is clear that the Enzymatic Reduction Method of Nitrate-N Analysis yields highly accurate and precise results. 3.4 Comparison of the Enzymatic Reduction Method to the Cadmium Reduction Method The samples matrices listed in Table 1 were also analyzed by the cadmium reduction method for Nitrate-N content (EPA Method 353.2). These results are compared to results from the Enzymatic Reduction Method in Table 10. For most of the matrices, the Enzymatic Reduction Method resulted in determination of a higher Nitrate-N content than the Cadmium Reduction Method, but two matrices were greater by CdR and two were the same for the two methods. Most of the matrices analyzed were within the range of the standard curves of the Methods (or diluted to bring them into range), but four matrices were below the lowest calibrant used to prepare the standard curves. These 4 matrices were WW-1, WW-4, WW-7, and SW-1, which are footnoted in Table 10. These matrices had RPD greater than 10%, which is not unexpected since the determined values are not really valid. One matrix with low nitrate content (WW-1) had an acceptable RPD. All the other matrices had RPD less than 10%. It is clear from the results in Table 10, that the Enzymatic Reduction Method gave comparable results to the certified EPA reference method. In addition, extensive studies comparing the Enzymatic Reduction Method and Cadmium Reduction Method for the Nitrate-N content of natural water samples at the USGS have shown the two methods yield comparable results (Patton et al., 2002; Patton and Kryskalla, 2011; 2013). A less extensive study comparing the two methods found no differences in variability meaning that the standard deviation of the analysis of analyte nitrate in double-distilled water were not significantly different (p < 0.05) (Ringuet et al., 2011). NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 8

12 Section 4 Validation Conclusions The USEPA requires for Tier 3 (nationwide) validation of an Alternate Test Procedure (ATP) Method that nine different laboratories analyze the analyte content of one sample of eight different wastewater matrices, and one sample matrix of ocean water (EPA, 1999). Thus, the ATP Method will be in compliance with the Clean Water Act (CWA) and validate the ATP Method for nationwide application as a regulatory method. For the Enzymatic Reduction Method for Nitrate-N Analysis, the requirements of Tier 3 validation were met by analysis of eight wastewater matrices and one seawater matrix by ten laboratories (see Table 1 for list of Wastewater Sample Matrices analyzed in this study and Tables 8 and 9 for details of the analysis by 9 of the 10 laboratories listed in Table 2). The spiking studies of the Wastewater Sample Matrices (spikes of 0.5 mg N/L analyzed six times) indicated that there was little or no matrix effect on the Method by the eight Wastewater matrices (Table 8 and 9). Seawater (ocean water) was also analyzed (Table 8 and 9). No significant effect of the seawater matrix spiked with 0.5 mg N/L nitrate and analyzed 6 times, was found on the Nitrate-N content determined for seawater (Table 8 and 9). Accuracy of the Method was shown to be very high by analysis of 3 reference standards (Table 10) and the precision of the Quality Control results (Tables 5 and 6). The MDL evaluation of the equipment used in the study demonstrated that the Method has a detection limit of less than mg N/L on the DA analyzers used in the study (Table 7). Few interfering substances, if any, were discovered in the present study. Previous analysis of interferences with specific compounds showed that there was little interference with the Method (Patton and Kryskalla, 2011; Patton and Kryskalla, 2013). See also data on interferences in The Method description in Appendix B. Comparison of the analysis of the eight wastewater and seawater matrices and three reference standards by the Enzymatic Reduction Method and the certified Cadmium Reduction Method (EPA Method 353.2) indicated that very similar results were obtained (Table 11). Previous studies have also found the two methods yielded similar results (Patton et al., 2002; Patton and Kryskalla, 2011; 2013; Ringuet et al., 2011). In summary, the Enzymatic Reduction Method for Nitrate-N Analysis has been validated by a robust Inter-Laboratory Study of Wastewater and Seawater Sample Matrices. All laboratories analyzing the Sample Matrices met all Quality Control criteria for valid analyses prior to analyzing the samples. The Calibration Curve, Nitrate Reduction Efficiency, and IPR/OPR recoveries (Tables 3, 4, 5, 6, and 7) were within the acceptable range before analyzing the unknown Sample Matrices. Analysis of certified Nitrate Standards indicated that the Method is highly accurate and capable of providing definitive analysis of Nitrate-N content of Wastewater NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 9

13 in the field. For Discrete Analyzers running the Method, the MDL ranged from to mg N/L (Table 7). Thus, the requirements of the Tier 3 level Alternate Test Procedure Protocol have been met for validation of the Enzymatic Reduction Method for Nitrate-N Analysis by the Inter-Laboratory Validation Study reported herein. NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 10

14 Section 5 References and Glossary Protocol for EPA Approval of Alternate Test Procedures for Organic and Inorganic Analytes in Wastewater and Drinking Water, USEPA, Campbell, Wilbur H. (1999) Nitrate Reductase Structure, Function and Regulation: Bridging the Gap between Biochemistry and Physiology, Annual Review of Plant Physiology and Plant Molecular Biology 50: Campbell, Wilbur H., P Song, GG Barbier (2006) Nitrate Reductase for Nitrate Analysis in Water. Environmental Chemistry Letters, 4: Patton CJ, AE Fischer, WH Campbell & ER Campbell (2002) Corn leaf nitrate reductase: A nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis. Environmental Science and Technology, 36: Patton, C.J., and Kryskalla, J.R., 2011, Colorimetric determination of nitrate plus nitrite in water by enzymatic reduction, automated discrete analyzer methods: U.S. Geological Survey Techniques and Methods, book 5, chap. B8, 34 p. (Available on line at Patton, C.J., and Kryskalla, J.R., 2013, Analytical properties of some commercially available nitrate reductase enzymes evaluated as replacements for cadmium in automated, semiautomated, and manual colorimetric methods for determination of nitrate plus nitrite in water, USGS Scientific Investigations Report ( Ringuet, R., L. Sassano, and Z. L. Johnson (2011) A Suite of microplate reader-based colorimetric methods to quantify ammonium, orthophosphate, and silicate concentrations for aquatic nutrient monitoring. Journal of Environmental Monitoring, 13: Glossary: DA = Automated Discrete Analyzer IPR = Initial Performance and Recovery OPR = Ongoing Performance and Recovery MDL = Method Detection Limit MS = Matrix Spike MSD = Matrix Spike Duplicate QA = Quality Assurance QC = Quality Control NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 11

15 Table 1. List of Sample Matrices for Analysis Sample Number Sample Type Matrix Identifier Filtered Acidified Waste Water Samples Denver area treatment plant Influent wastewater Denver area treatment plant Wastewater effluent #1 Denver area treatment plant Wastewater effluent #2 Michigan paper mill waste stream effluent Denver area metal finisher waste stream effluent Denver area Commercial laundry waste stream effluent Environmental Resources Associates #507 Hardness WasteWatR reference material Michigan Confined Animal Feeding Operation (CAFO) effluent from tiled field WW-1 Yes Yes WW-2 Yes Yes WW-3 Yes Yes WW-4 Yes Yes WW-5 Yes Yes WW-6 Yes Yes WW-7 Yes Yes WW-8 Yes Yes Other Sample 9 Low-nutrient seawater (collected offshore Hawaii) SW-1 Yes No Reference Standards ERA # 608 Reference Standard SR-1 Yes Yes USGS PE N-116 (low nutrient-fortified river water) USGS PE N-115 (high nutrient-fortified river water) SRM-1 Yes No SRM-2 Yes No NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 12

16 Table 2 Participant Laboratories Lab Number Lab Name Contact Analytical Method 2 USGS/NWQL Charles Patton Discrete Analyzer #1 3 USGS/NWQL Charles Patton Discrete Analyzer #2 4 OI Analytical William Lipps Discrete Analyzer 5 ThermoFisher Stephen White Discrete Analyzer #1 6* ThermoFisher Stephen White Discrete Analyzer #2 7 ThermoFisher Stephen White Discrete Analyzer #3 8 Univ. of Maryland/Solomons Jerry Frank Discrete Analyzer 9 Klamath Tribes Kris Fischer Discrete Analyzer 10 Geochemical Testing Tim Boergstresser Discrete Analyzer 11 Unity/Westco Scientific Bill Georgian Discrete Analyzer 12 Astoria-Pacific Winston Pavitt, CEO Discrete Analyzer *Laboratory 6 dropped out of the study. Laboratory 1 employed a different analytical procedure than that used by the other laboratories (Lab Numbers 2-12). Therefore, those data have not been included as part of this final study report. NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 13

17 Table 3 Calibration of Enzymatic Reduction Method Summary A. Statistics for the Calibration Curve for Each Laboratory NaR Method Slope A-540 nm Correlation Coefficient A-540 per mg N/L Intercept r 2 Lab 2 DA Lab 3 DA Lab 4 DA Lab 5 DA Lab 7 DA (0.9999)ꜟ Lab 8 DA Lab 9 DA Lab 10 DA (0.9991) ꜟ Lab 11* DA Lab 12 DA *Lab 11 used 550 nm; ꜟPolynomial Fit. B. Calibration Nitrate Concentrations (mg N/L) used. Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 10 Lab NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 14

18 Table 4 Enzymatic Reduction Efficiency Summary Acceptance Standard is 90% or greater reduction efficiency. 2nd Source = Nitrate Standard in mg N/L and Nitrite Standard = mg N/L A-540 nm mg N/L Reduction Efficiency Lab 2 2nd Source Nitrite Standard % Lab 3 2nd Source Nitrite Standard % Lab 4 2nd Source Nitrite Standard % Lab 5 2nd Source Nitrite Standard % Lab 7 2nd Source Nitrite Standard % Lab 8 2nd Source Nitrite Standard % Lab 9 2nd Source Nitrite Standard % Lab 10 2nd Source Nitrite Standard % Lab 11* 2nd Source % Nitrite Standard Lab 12 2nd Source Nitrite Standard % *Lab 11 used 550 nm NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 15

19 Lab Table 5 Initial Performance and Recovery (IPR) Summary # Analyses Spike Conc. (mg/l) Mean Recovery (%) RSD (%) Minimum Recovery (%) Maximum Recovery (%) NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 16

20 Lab Table 6 Ongoing Performance and Recovery (OPR) Summary OPR mg N/L # Analyses* RSD = Relative Standard Deviation Mean Recovery % RSD % Minimum Recovery (%) Maximum Recovery (%) ** ** *Some Labs did more OPR than are summarized here for reasons of repeated analyses. ** Lab 4 initially used OPR = 2.50 mg N/L and then switched to OPR = 2.00 mg N/L. NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 17

21 Table 7 Method Detection Limit (MDL) Summary Abbreviations: DA = Discrete Analyzer; NA = Not Analyzed. Lab Method MDL Replicates Spike Spike/MDL mg N/L mg N/L Ratio 2 DA DA (7)* DA DA DA DA DA DA NA 11 DA DA *One replicate discarded due to issue with blank NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 18

22 Table 8 Nitrate-N Content and Spike Analysis of Waste Water and Seawater Matrices Abbreviations: RSD = Relative Standard Deviation; RPD = Relative Percent Difference Laboratory Matrix Nitrate-N (mg N/L) Unspiked Sample Summary Standard Deviation (mg N/L) RSD (%) Dilution Final Nitrate-N (mg N/L) 2 WW X WW X WW None WW X WW None WW None WW X WW None SW None Spiked Sample Summary Mean Spike 2 % Recovery (%RPD) Mean Spike 1 % Recovery (%RPD) (1.0138) (0.8002) (4.7179) (2.4315) (2.4348) (0.3376) (0.1534) (1.5385) (2.4635) (0.7319) (0.8933) (4.5016) (1.0010) (3.5211) (2.2096) (0.0768) (9.6154) (1.9760) Mean Spike 3 % Recovery (%RPD) (0.6115) (0.3202) (0.6543) (0.5755) (1.2693) (1.3639) (1.6296) (1.5968) (1.1698) NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 19

23 Table 9 Detailed Data Supporting Table 8 Data in this table are summarized from each Laboratory's Summary Excel File in Appendix F. Standard Curve Data are in Table 3. Spiking was done at 0.5 mg N/L Matrix ID Lab Dilution Factor WW WW-1 MS WW-1 MSD A-540 Nitrate-N Nitrate-N Spike 1 Spike 2 Spike 3 Spiked Spike Spike Measured Final Spike (mg- Recovery Spike Recovery Spike Spiked (mg-n/l) (mg-n/l) A-540 N/L) % A-540 % A-540 Matrix Replicates WW-1 Mean Spike Recovery (%) Relative Percent Difference (%) Lab A-550 WW WW-2 MS WW-2 MSD WW-2 Mean Spike Recovery (%) Relative Percent Difference (%) Lab A-540 WW WW-3 MS WW-3 MSD WW-3 Mean Spike Recovery (%) Relative Percent Difference (%) Lab A-540 WW WW-4 MS WW-4 MSD WW-4 Mean Spike Recovery (%) Relative Percent Difference (%) Lab A-540 WW WW-5 Mean Spike Recovery (%) Relative Percent Difference (%) Lab A-540 WW WW-6 MS WW-6 MSD WW-6 Mean Spike Recovery (%) Relative Percent Difference (%) Lab A-540 WW WW-7 MS WW-7 MSD WW-5 MS WW-5 MSD WW-7 Mean Spike Recovery (%) Relative Percent Difference (%) Lab A-540 WW WW-8 MS WW-8 MSD WW SW-1 Lab SW-1 MS SW-1 MSD SW Mean Spike Recovery (%) Relative Percent Difference (%) A Mean Spike Recovery (%) Relative Percent Difference (%) Spiked (mg-n/l) Spike (mg-n/l) Recovery % NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 20

24 Table 10 Standard Reference Materials Summary Labs 5 and 8 did not analyze these matrices. Sample Matrix ERA #698 - Finished drinking water (Target value from ERA) # Concentration Result Sample Target Lab Recovery (%) RSD Analyses (mg N/L) mg N/L Mean Min. Max Mean Min. Max % ERA 6.80 # ±0.12 (1.82%) N = 38 Mean = ± 0.62% Mean Recovery = 98.20% Sample Matrix SRM-1 - USGS PE Sample N-116 (Target Most Probable Value from USGS*) # Concentration Result Sample Target Lab Recovery (%) RSD Analyses (mg N/L) mg N/L Mean Min. Max Mean Min. Max % SRM ±0.011 (2.44%) N = 36 Mean = ± 2.34% Mean Recovery = 99.62% NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 21

25 Table 10 Continued Sample Matrix SRM-2 - USGS PE Sample N-115 (Target Most Probable Value from USGS*) Sample Target Lab # Analyses Concentration Result (mg N/L) Recovery (%) RSD mg N/L Mean Min. Max Mean Min. Max % SRM ±0.009 (0.38%) N = 25 Mean = ± 3.02% Mean Recovery = 99.43% *Data from US Geological Survey (see link below and Appendix D of the Final Report) ( For SRM-1, which is USGS N-116, 25 analysis were reported by colorimetric methods with the following results (units = mg N/L): MPV = 0.443; Fps = (Fps = StDev/1.4826) therefore, StDev = (2.44%) For SRM-2, which is USGS N-115, 25 analysis were reported by colorimetric methods with the following results: MPV = 0.230; Fps = 0.013; StDev = (0.38%) MPV = Most Probable Value; Fps = F-pseudo-sigma or MAD = Median Absolute Deviation NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 22

26 Table 11 Comparison of Nitrate-N Content of Sample Matrices and Standard References Determined by Discrete Analyzer Enzymatic Reduction Method (NaR) and Cadmium Reduction Air-Segmented Continuous Flow Analyzer Method (CdR) Abbreviations: RPD = Relative Percent Difference for NaR-CdR (+ = NaR higher; - = CdR higher) Note: NaR and CdR Method were done by Laboratory 2 on 19Jul2013 and values are mean of 3 replicates. Sample Matrix NaR CdR RPD mg N/L mg N/L % WW-1* WW WW WW-4* WW WW WW-7* WW SW-1* SR SRM SRM *These values at or below the lowest calibrant of the method standard curve. NECi Enzymatic Reduction Method for Nitrate ATP Validation Report Page 23